Structural protein expression and phenotypic properties of the mutant viruses. (A) Northern blot analysis of mutant RNAs isolated at 48 hours post transfection from MARC-145 cells transfected with WT, EX, S-LL, S-RR and S-RL plasmids. (B) PRRSV N protein expression of WT and mutants in transfected cells. Expression of N protein was visualized by immunofluorescence staining with anti-N antibody at 24 hours post-transfection. (C) Viral plaque morphology assay. 0.01 MOI of P1 supernatants were inoculated in fresh MARC-145 cells and covered by MEM containing 2% FBS and 1% low melting agarose, and the plaques were visualized at 5 days post infection by crystal violet staining. The plaque sizes of the WT (■) and mutant EX (▲), L-LL(*), L-RR(▏), L-RL(╳), and S-RL (+), were measured by a millimeter ruler after monolayers were stained with crystal violet. The bars represent the average plaque diameters. (D) Viral multi-step growth curves. MARC-145 cells infected at an MOI of 0.01 with the P1 passage parental virus and mutant viruses and harvested at the indicated time points. The virus titers were determined by plaque assay and the results were mean values from three independent experiments. Viral titers were expressed as log10 PFU/ml.